A doença granulomatosa crônica é uma imunodeficiência primária dos fagócitos causada por mutações no sistema NADPH oxidase resultando em burst oxidativo ausente ou reduzido. Nosso objetivo foi realizar uma análise genética molecular do complexo NADPH oxidase em pacientes com diagnóstico clínico de DGC. Cinqüenta e quatro pacientes com diagnóstico clínico sugestivo da DGC foram incluídos em nosso estudo. As populações de neutrófilos e monócitos foram avaliadas pela capacidade de produzir peróxido de hidrogênio por meio do teste de DHR. Dezoito pacientes apresentaram defeito no burst oxidativo, enquanto trinta e oito apresentaram produção de peróxido normal. O DNA genômico dos dezoito pacientes com burst oxidativo diminuído foi extraído, os genes da cadeia beta polipeptídica do complexo citocromo b e o factor citoplasmático de neutrófilos, foram sequenciados. Sete pacientes apresentaram diferentes mutações, tanto no gene CYBB como no NCF1. Concluímos que a combinação do teste de DHR e o sequenciamento direto são métodos eficazes para o diagnóstico genético da DGC. / Chronic granulomatous disease is a primary immunodeficiency caused by mutations in the phagocyte NADPH oxidase system resulting in absent or reduced oxidative burst. Our goal was to perform a molecular genetic analysis of complex NADPH oxidase in patients with clinical diagnosis of CGD. Fifty-four patients with a clinical diagnosis of CGD were included in our study. The populations of neutrophils and monocytes were evaluated for the ability to produce hydrogen peroxide through the DHR test. Eighteen patients had a defect in the oxidative burst, while thirty-eight had normal peroxide production. Genomic DNA of the eighteen patients with decreased oxidative burst was extracted, the genes the chain complex cytochrome beta polypeptide and the neutrophil cytoplasmic factor, were sequenced. Seven patients had different mutations, both in the CYBB gene as in NCF1. We conclude that the combination of direct sequencing and DHR test methods are effective for the genetic diagnosis of CGD.

A doença granulomatosa crônica é uma imunodeficiência primária dos fagócitos causada por mutações no sistema NADPH oxidase resultando em burst oxidativo ausente ou reduzido. Nosso objetivo foi realizar uma análise genética molecular do complexo NADPH oxidase em pacientes com diagnóstico clínico de DGC. Cinqüenta e quatro pacientes com diagnóstico clínico sugestivo da DGC foram incluídos em nosso estudo. As populações de neutrófilos e monócitos foram avaliadas pela capacidade de produzir peróxido de hidrogênio por meio do teste de DHR. Dezoito pacientes apresentaram defeito no burst oxidativo, enquanto trinta e oito apresentaram produção de peróxido normal. O DNA genômico dos dezoito pacientes com burst oxidativo diminuído foi extraído, os genes da cadeia beta polipeptídica do complexo citocromo b e o factor citoplasmático de neutrófilos, foram sequenciados. Sete pacientes apresentaram diferentes mutações, tanto no gene CYBB como no NCF1. Concluímos que a combinação do teste de DHR e o sequenciamento direto são métodos eficazes para o diagnóstico genético da DGC. / Chronic granulomatous disease is a primary immunodeficiency caused by mutations in the phagocyte NADPH oxidase system resulting in absent or reduced oxidative burst. Our goal was to perform a molecular genetic analysis of complex NADPH oxidase in patients with clinical diagnosis of CGD. Fifty-four patients with a clinical diagnosis of CGD were included in our study. The populations of neutrophils and monocytes were evaluated for the ability to produce hydrogen peroxide through the DHR test. Eighteen patients had a defect in the oxidative burst, while thirty-eight had normal peroxide production. Genomic DNA of the eighteen patients with decreased oxidative burst was extracted, the genes the chain complex cytochrome beta polypeptide and the neutrophil cytoplasmic factor, were sequenced. Seven patients had different mutations, both in the CYBB gene as in NCF1. We conclude that the combination of direct sequencing and DHR test methods are effective for the genetic diagnosis of CGD.

Chronic granulomatous disease (CGD) is associated with defective function of the NADPH-oxidase system in conjunction with phagocytic defects which leads to granuloma formation and serious infectious complications. This is often associated with significant morbidity and mortality. The association of defective phagocyte function with other coincidental immune defects is unknown. Defects in innate pathways seen with CGD, including complement systems, and toll-like and dectin receptor pathways, have not been described before. We present the case of a 2-year old male patient hospitalized with recurrent pneumonia, a non-healing skin ulcer, necrotizing lung granulomas, and epididymo-orchitis. Defective neutrophil chemiluminescence was detected by dihydrorhodamine (DHR) testing. Further evaluation demonstrated characteristic molecular mutations of CYBB consistent with CGD. Immune evaluation demonstrated polyclonal hyperglobulinemia, but a greatly reduced mannose binding lectin (MBL) level. Six biallelic polymorphisms in MBL gene and its promoter were analyzed using Light Cycler™ Real-time PCR assay. The LXPA/LYPB haplotype of MBL was detected in our patient; the latter is the defective haplotype associated with low MBL levels. Due to the implications for innate immunity and the protection against bacterial, viral, and fungal infections provided by MBL, a deficiency of this protein may have disastrous consequences on the long term outcomes of CGD. MBL deficiency can also complicate other disorders affecting the immune system, significantly increasing the risk of infection in such patients. Further studies looking at the frequency and implications of MBL deficiency in CGD are needed. © 2012 Bentham Science Publishers.

Os fagócitos contêm uma nicotinamida adenina dinucleotídeo fosfato (NADPH) oxidase associada à membrana, que gera superóxido e outros reativos intermediários do oxigênio. Defeitos nesta oxidase em seres humanos resultam na doença granulomatosa crônica (DGC). Mutações próximas aos sítios de splicing que interferem com o processamento do RNA mensageiro, acarretando deleção de um ou mais exons, são cada vez mais freqüentes na literatura científica, nesses casos, os mecanismos moleculares que levam a DGC nem sempre são totalmente esclarecidos, assim como o efeito do IFN-g, seja sobre o processamento da mensagem ou estabilidade dos transcritos. Com base nessas informações o objetivo geral deste trabalho é investigar o efeito do IFN-g sobre a regulação do sistema NADPH oxidase fagocítico humano. Através dos resultados obtidos, não se pode constatar melhora na produção de ânions superóxido após o tratamento com IFN-g em pacientes com defeito de splicing, no entanto detectou-se aumento da expressão do gene CYBB através de PCR convencional e através de real-time PCR além de um aumento na marcação de proteínas do spliceossoma através do FAN. / Phagocytes have a nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) associated to plasmatic membrane that generates superoxide and other oxygen reactive intermediates. Defects on this oxidase in humans result in a disorder called Chronic Granulomatous Disease (CGD). Mutations next to splicing sites that interfere with the mRNA processing leading to deletion of one or more exons, are even more frequent on scientific literature, in these cases, the molecular mechanisms causing CGD are not always completetly clear, as well as the effect of IFN-g on the mRNA processing or on the stability of transcripts. Based on this information our aim is to investigate the effect of IFN-g on the regulation of the human NADPH oxidase phagocyte system.. With the obtained results it wasnt possible to see an increase in anion superoxide production after the IFN-g treatment in patients with splicing defects, however it was detected an increase on the expression of CYBB gene by conventional and real-time PCR besides an increase in the marking of spliceossomal proteins by FAN.

O sistema NADPH oxidase humano é responsável pela geração de reativos intermediários do oxigênio e defeitos neste sistema resultam na Doença Granulomatosa Crônica (DGC). Nesta tese de doutorado, investigamos o efeito do IFN-g sobre eventos pós-transcricionais em pacientes com DGC ligada ao X, ocasionada por defeitos de splicing. Os dados obtidos sugerem que o uso do IFN-g in vitro interfere no processamento da mensagem causando aumento da expressão de transcritos do gene CYBB e NCF1 em células B-EBV de indivíduos sadios e pacientes DGC analisados. Observamos também que o IFN-g dimunui a expressão dos genes THOC4 NONO, SF3A1, SRRM1 e UPF3A e promove aumento de expressão de SRSF10, SNRPA1 e C2 em células B-EBV de paciente X-DGC secundária a defeitos de splicing. Identificamos que o IFN-g e o TNF-α aumentam a expressão das proteínas envolvidas no processo do splicing. Concluímos que o IFN-g aumenta a expressão de genes importantes para uma resposta eficiente do sistema imunológico, incluindo os do sistema NADPH oxidase, além de promover aumento da expressão de genes e de proteínas relacionados ao processo de splicing, que podem estar relacionados aos efeitos benéficos observados no uso do IFN-g em pacientes com DGC ligada ao X, ocasionada por defeitos de splicing. / The human phagocyte NADPH oxidase is responsible for the generation of reactive oxygen intermediates and defects in this system result in Chronic Granulomatous Disease (CGD). In this PhD Thesis, we investigated the effect of IFN-g on post-transcriptional events in normal individuals and patients with X-linked CGD, caused by splicing defects. The obtained data suggests that the use of IFN-g in vitro interferes in the message processing causing an increase of expression of CYBB and NCF1 gene transcripts in B-EBV cells of healthy individuals and analyzed CGD patients. We also observed that IFN-g decreases the expression of THOC4, NONO, SF3A1, SRRM1 and UPF3A, and increases the expression of SRSF10, SNRPA1 and C2 genes in cells from X-CGD patients, due to splicing defects. We identified that IFN-g and TNF-α induce expression of proteins involved in the splicing process. We conclude that IFN-g increases the expression of important genes for an effective immune response, including the NADPH oxidase system genes, and promotes augment of gene and protein expression related to the splicing process, which may be related to the beneficial effects related to the use of IFN-g in CGD patient caused by splicing defects.

La Granulomatose Septique Chronique (CGD) est une maladie génétique rare de l’immunodéficience innée affectant les cellules phagocytaires (neutrophiles, macrophages). Elle est causée par des mutations dans les sous-unités du complexe NADPH oxydase formé du cytochrome b558 membranaire (NOX2 associé à p22phox) et de facteurs cytosoliques (p47phox, p67phox et p40phox). La déficience de ce complexe enzymatique va conduire à l’absence de formation de formes réactives de l’oxygène (FRO) microbicides et donc à l’apparition d’infections graves et récurrentes très tôt dans l’enfance. La chimioprophylaxie à vie permet de protéger ces patients mais peut être responsable d’effets indésirables. La seule thérapie curative est la transplantation de moelle osseuse mais tous les patients ne peuvent en bénéficier, et la thérapie génique n’est pas encore envisageable. Il y a donc un manque réel de nouvelles thérapies pour cette maladie. Cependant pour développer de nouveaux traitements, il faut disposer de modèles physiopathologiques pertinents. Or, les modèles existants sont imparfaits ou manquants. Le but de notre travail est donc de produire des modèles cellulaires et animaux de la CGD pour développer dans un second temps, une nouvelle approche thérapeutique basée sur l’utilisation de protéoliposomes.Grâce à leurs propriétés de pluripotence et d’auto-renouvellement à l’infini, les cellules souches pluripotentes induites (iPS) sont un outil puissant pour la modélisation physiopathologique. Ainsi, à partir de fibroblastes de patients atteints de CGD reprogrammés en cellules iPS, nous avons mis au point un protocole efficace de différenciation hématopoïétique in vitro en neutrophiles et macrophages. Nous avons montré que ces cellules phagocytaires sont matures et reproduisent parfaitement le phénotype déficient en FRO des patients CGD. Nous avons donc obtenu des modèles cellulaires pertinent modélisant trois formes génétiques de CGD, la CGD liée à l’X et deux formes autosomiques récessives, CGDAR22 et CGDAR47.Nous avons ensuite réalisé la preuve du concept de l’efficacité de protéoliposomes thérapeutiques sur les macrophages modélisés de la forme CGDX, la forme génétique la plus fréquente (70 % des cas) due à l’absence du cytochrome b558 membranaire (NOX2/p22phox). Grâce à une collaboration avec la start-up Synthelis SAS, des liposomes contenant le cytochrome b558 au niveau de la membrane lipidique ont été produits dans un système d’expression acellulaire basé sur l’utilisation d’extraits d’Escherichia coli. Ces liposomes NOX2/p22phox sont capables de reconstituer une enzyme NADPH oxydase fonctionnelle in vitro et de délivrer le cytochrome b558 à la membrane plasmique des macrophages CGDX qui présentent alors une restauration de l’activité NADPH oxydase avec la production de FRO.Enfin, nous nous sommes proposés de générer des souris dites « humanisées » par transplantation de cellules souches hématopoïétiques CD34+ capables de prise de greffe et de reconstitution hématopoïétique dans des souris immunodéficientes. A partir de cellules iPS saines, nous avons réussi à produire des cellules hématopoïétiques CD34+ possédant un potentiel hématopoïétique in vitro. Cependant, malgré des résultats encourageants, aucune prise de greffe in vivo n’a pu être réellement confirmée à ce jour.Pour conclure, nous avons donc montré au cours de ce projet, la production de modèles cellulaires de trois formes génétiques de CGD à partir de cellules iPS. Puis le modèle de macrophages CGDX nous a permis de faire la preuve de l’efficacité d’une nouvelle thérapie in vitro, une « enzymothérapie substitutive liposomale », qui pourrait à terme, offrir une alternative thérapeutique pour le traitement des infections aigües pulmonaires des patients CGD réfractaires aux traitements antibiotiques et antifongiques conventionnels. / Chronic Granulomatous Disease (CGD) is a rare inherited pathology of the innate immune system that affects the phagocytic cells (neutrophils, macrophages). This disease is caused by mutations in the subunits of the NADPH oxidase complex composed of the membrane cytochrome b558 (NOX2 associated with p22phox) and the cytosolic components (p47phox, p67phox et p40phox). Dysfunction in this enzymatic complex leads to the absence of microbicidal reactive oxygen species (ROS) and therefore to the development of recurrent and life-threatening infections in early childhood. Life-long prophylaxis is used to protect these patients but it may be responsible for side effects. Bone marrow transplantation is the only curative treatment but it can not be proposed to all the patients. In addition, gene therapy is not possible up to now. So there is a real lack of new therapies for this disease. However, to develop new therapeutic approaches, relevant physiopathological models must be available. Actually, existing models are imperfect or missing. Thus, the goal of our work is to produce cellular and animal models of CGD to develop a new proteoliposome-based therapy.Induced pluripotent stem cells (iPSCs) are a powerful tool for physiopathologic modeling due to their pluripotency and self-renewal properties. Using CGD patient-specific iPSCs regrogrammed from fibroblasts, we developped an efficient protocol for in vitro hematopoietic differentiation into neutrophils and macrophages. We showed that the phagocytic cells produced are mature and reproduce the ROS-deficient phenotype found in CGD patients. Thus, we obtained relevant cellular models for three genetic forms of CGD: X-linked CGD and the two autosomal recessive forms AR22CGD and AR47CGD.Then, we demonstrated the proof-of-concept of the efficacy of therapeutic proteoliposomes on X-CGD iPS-derived macrophages. Indeed, X-CGD is the main form of the disease (70% of cases) and is caused by the absence of the membrane cytochrome b558 (NOX2/p22phox). Thanks to a collaboration with the start-up Synthelis SAS, liposomes integrating the cytochrome b558 into lipid bilayers were produced in an E. coli-based cell-free protein expression system. These NOX2/p22phox liposomes were able to reconstitute a functional NADPH oxidase enzyme in vitro and to deliver the cytochrome b558 at the plasma membrane of X-CGD macrophages, leading to restore the NADPH oxidase activity with a ROS production.Finally, we proposed to generate « humanized » mice models with a human immune system after transplantation of CD34+ hematopoietic stem cells able to engraft and reconstitute long-term hematopoiesis in immunodeficient mice. Using healthy iPSCs, we successfuly produced CD34+ hematopoietic cells with in vitro hematopoietic potential. However, no in vivo engraftment was really confirmed yet.In conclusion, during this project, we produced cellular models of three genetic forms of CGD using patient-specific iPSCs. Then, X-CGD macrophages were used to demonstrate in vitro the efficacy of a new therapy. This « liposomal replacement enzymotherapy » could, in the future, represents a curative alternative against life-threatening lung infections refractory to conventional antibiotic and antifungal therapy.

Os fagócitos contêm uma nicotinamida adenina dinucleotídeo fosfato (NADPH) oxidase associada à membrana, que gera superóxido e outros reativos intermediários do oxigênio. Defeitos nesta oxidase em seres humanos resultam na doença granulomatosa crônica (DGC). Mutações próximas aos sítios de splicing que interferem com o processamento do RNA mensageiro, acarretando deleção de um ou mais exons, são cada vez mais freqüentes na literatura científica, nesses casos, os mecanismos moleculares que levam a DGC nem sempre são totalmente esclarecidos, assim como o efeito do IFN-g, seja sobre o processamento da mensagem ou estabilidade dos transcritos. Com base nessas informações o objetivo geral deste trabalho é investigar o efeito do IFN-g sobre a regulação do sistema NADPH oxidase fagocítico humano. Através dos resultados obtidos, não se pode constatar melhora na produção de ânions superóxido após o tratamento com IFN-g em pacientes com defeito de splicing, no entanto detectou-se aumento da expressão do gene CYBB através de PCR convencional e através de real-time PCR além de um aumento na marcação de proteínas do spliceossoma através do FAN. / Phagocytes have a nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) associated to plasmatic membrane that generates superoxide and other oxygen reactive intermediates. Defects on this oxidase in humans result in a disorder called Chronic Granulomatous Disease (CGD). Mutations next to splicing sites that interfere with the mRNA processing leading to deletion of one or more exons, are even more frequent on scientific literature, in these cases, the molecular mechanisms causing CGD are not always completetly clear, as well as the effect of IFN-g on the mRNA processing or on the stability of transcripts. Based on this information our aim is to investigate the effect of IFN-g on the regulation of the human NADPH oxidase phagocyte system.. With the obtained results it wasnt possible to see an increase in anion superoxide production after the IFN-g treatment in patients with splicing defects, however it was detected an increase on the expression of CYBB gene by conventional and real-time PCR besides an increase in the marking of spliceossomal proteins by FAN.

O sistema NADPH oxidase humano é responsável pela geração de reativos intermediários do oxigênio e defeitos neste sistema resultam na Doença Granulomatosa Crônica (DGC). Nesta tese de doutorado, investigamos o efeito do IFN-g sobre eventos pós-transcricionais em pacientes com DGC ligada ao X, ocasionada por defeitos de splicing. Os dados obtidos sugerem que o uso do IFN-g in vitro interfere no processamento da mensagem causando aumento da expressão de transcritos do gene CYBB e NCF1 em células B-EBV de indivíduos sadios e pacientes DGC analisados. Observamos também que o IFN-g dimunui a expressão dos genes THOC4 NONO, SF3A1, SRRM1 e UPF3A e promove aumento de expressão de SRSF10, SNRPA1 e C2 em células B-EBV de paciente X-DGC secundária a defeitos de splicing. Identificamos que o IFN-g e o TNF-α aumentam a expressão das proteínas envolvidas no processo do splicing. Concluímos que o IFN-g aumenta a expressão de genes importantes para uma resposta eficiente do sistema imunológico, incluindo os do sistema NADPH oxidase, além de promover aumento da expressão de genes e de proteínas relacionados ao processo de splicing, que podem estar relacionados aos efeitos benéficos observados no uso do IFN-g em pacientes com DGC ligada ao X, ocasionada por defeitos de splicing. / The human phagocyte NADPH oxidase is responsible for the generation of reactive oxygen intermediates and defects in this system result in Chronic Granulomatous Disease (CGD). In this PhD Thesis, we investigated the effect of IFN-g on post-transcriptional events in normal individuals and patients with X-linked CGD, caused by splicing defects. The obtained data suggests that the use of IFN-g in vitro interferes in the message processing causing an increase of expression of CYBB and NCF1 gene transcripts in B-EBV cells of healthy individuals and analyzed CGD patients. We also observed that IFN-g decreases the expression of THOC4, NONO, SF3A1, SRRM1 and UPF3A, and increases the expression of SRSF10, SNRPA1 and C2 genes in cells from X-CGD patients, due to splicing defects. We identified that IFN-g and TNF-α induce expression of proteins involved in the splicing process. We conclude that IFN-g increases the expression of important genes for an effective immune response, including the NADPH oxidase system genes, and promotes augment of gene and protein expression related to the splicing process, which may be related to the beneficial effects related to the use of IFN-g in CGD patient caused by splicing defects.

Les cellules Natural Killer (NK) représentent une population de cellules innées lymphoïdes aux fonctions anti-infectieuses et antitumorales. Les leucémies aiguës lymphoblastiques pré-B (LAL pré-B) constituent le cancer de l’enfant le plus fréquent et ont été décrites comme résistantes à la cytotoxicité médiée par les NK bien que les bases moléculaires demeurent inconnues.L’objectif de ces travaux a été de caractériser cette résistance. En développant un essai de cytotoxicité par cytométrie en flux et en utilisant des cellules effectrices activées in vitro, nous avons établi la sensibilité retardée des LAL pré-B à la cytotoxicité NK : initialement résistantes après 4h d’incubation, elles sont fortement tuées après 25h.Cette cytotoxicité est contact-dépendante mais ni la voie de l’exocytose des granules cytotoxiques ni celle des récepteurs de mort n’y contribuent. La mort cellulaire des cibles est de profil apoptotique mais indépendante des caspases ; la signalisation mitochondriale l’amplifie partiellement. Interférer avec les dérivés de l’oxygène par un antioxydant diminue la cytotoxicité. Nous montrons que les cellules NK de patients atteints de granulomatose septique chronique liée à l’X présentent un défaut de cette nouvelle cytotoxicité. Nous démontrons l’expression par les NK des composants clefs d’une NADPH oxydase distincte du complexe utilisé par les phagocytes. Nos travaux établissent l’existence d’une voie de cytotoxicité non conventionnelle et en définissent les principaux prérequis moléculaires. / Natural Killer (NK) cells are innate lymphoid cells with anti-infectious and anti-tumoral activities. Among neoplasia, pre-B acute lymphoblastic leukemias (pre-B ALL) represent the most common form of cancer in childhood and were shown to be resistant to NK cell mediated cytotoxicity although the mechanisms explaining this phenomenon are incompletely understood.In the present work, we investigated the relative immune resistance of pediatric pre-B ALL targets to activated NK cells. We developed a flow cytometry based cytotoxicity assay to assess the NK activity and the involvement of long term cytotoxic pathways. Although pre-B ALL blasts were strongly resistant at 4h, we found a considerable delayed NK killing at 25h.Further investigations revealed that cell contact was mandatory for efficient killing but also that neither the granule exocytosis nor the death receptor pathway were involved. Target cell death was caspase independent but mitochondria signaling amplified it. We then showed that NK cells from patients with X-linked chronic granulomatous disease could not kill efficiently ALL blasts and that NK cells expressed key components of a NADPH oxidase complex that was distinct from the phagocyte type. Our work reveals an uncharacterized effector pathway among cytotoxic lymphocytes and establishes key molecular requirements for this unconventional pathway.

Chronic granulomatous disease (CGD) is associated with defective function of the NADPH-oxidase system in conjunction with phagocytic defects which leads to granuloma formation and serious infectious complications. This is often associated with significant morbidity and mortality. The association of defective phagocyte function with other coincidental immune defects is unknown. Defects in innate pathways seen with CGD, including complement systems, and toll-like and dectin receptor pathways, have not been described before. We present the case of a 2-year old male patient hospitalized with recurrent pneumonia, a non-healing skin ulcer, necrotizing lung granulomas, and epididymo-orchitis. Defective neutrophil chemiluminescence was detected by dihydrorhodamine (DHR) testing. Further evaluation demonstrated characteristic molecular mutations of CYBB consistent with CGD. Immune evaluation demonstrated polyclonal hyperglobulinemia, but a greatly reduced mannose binding lectin (MBL) level. Six biallelic polymorphisms in MBL gene and its promoter were analyzed using Light Cycler™ Real-time PCR assay. The LXPA/LYPB haplotype of MBL was detected in our patient; the latter is the defective haplotype associated with low MBL levels. Due to the implications for innate immunity and the protection against bacterial, viral, and fungal infections provided by MBL, a deficiency of this protein may have disastrous consequences on the long term outcomes of CGD. MBL deficiency can also complicate other disorders affecting the immune system, significantly increasing the risk of infection in such patients. Further studies looking at the frequency and implications of MBL deficiency in CGD are needed.